A variety of analytical techniques are used to characterize interactions between molecules, particularly in the context of assays directed to the detection and interaction of biomolecules. For example, antibody-antigen interactions are of fundamental importance in many fields, including biology, immunology and pharmacology. In this context, many analytical techniques involve binding of a "ligand" (such as an antibody) to a solid support, followed by contacting the ligand with an "analyte" (such as an antigen). Following contact of the ligand and analyte, some characteristic is measured which is indicative of the interaction, such as the ability of the ligand to bind the analyte. After measurement of the interaction, the ligand-analyte pair must be disrupted in order to "regenerate" free ligand for a further analytical measurement.
A number of techniques have been employed to regenerate surface-bound ligands. Most commonly, regeneration involves a series of trial and error attempts to remove the analyte from the ligand, while minimizing loss of ligand from the solid support. Care must also be taken not to use a regeneration solution that is too aggressive in order to avoid partial or complete loss of ligand activity. Furthermore, regeneration must not influence the ligand with regard to subsequent measurements, otherwise results from assay-to-assay will not be truly comparable. These problems may be avoided by simply discarding the solid support after each assay. However, this is undesirable since generation of the solid support having bound ligand can be both costly and time consuming, and very often the researcher has only limited quantities of the ligand and/or solid support. Accordingly, improved techniques for regenerating such surfaces are desired.
The need to effectively regenerate a solid surface may be illustrated in the context of biosensors which use surface plasmon resonance (SPR) to monitor the interactions between an analyte and a ligand bound to a solid support. In this regard, a representative class of biosensor instrumentation is sold by Biacore AB (Uppsala, Sweden) under the trade name BIAcore.RTM. (hereinafter referred to as "the BIAcore instrument"). The BIAcore instrument includes a light emitting diode, a sensor chip covered with a thin gold film, an integrated fluid cartridge and photo detector. Incoming light from the diode is reflected in the gold film and detected by the photo detector. At a certain angle of incidence ("the SPR angle"), a surface plasmon wave is set up in the gold layer, which is detected as an intensity loss or "dip" in the reflected light.
The SPR angle depends on the refractive index of the medium close to the gold layer. In the BIAcore instrument, dextran is typically coupled to the gold surface, and a ligand is bound to the dextran layer. The analyte of interest is injected in solution form onto the sensor surface through a fluid cartridge. Since the refractive index in the proximity of the gold film depends upon (1) the refractive index of the solution (which is constant) and, (2) the amount of material bound to the surface, the interaction between the bound ligand and analyte can be monitored as a function of the change in SPR angle.
A typical output from the BIAcore instrument is a "sensorgram," which is a plot of response (measured in "resonance units" or "RU") as a function of time. An increase of 1000 RU corresponds to an increase of mass on the sensor surface of approximately 1 ng/mm.sup.2. As sample containing an analyte contacts the sensor surface, the ligand bound to the sensor surface interacts with the analyte in a step referred to as "association." This step is indicated on the sensorgram by an increase in RU as the sample is initially brought into contact with the sensor surface. Conversely, "dissociation" normally occurs when sample flow is replaced by, for example, a buffer flow. This step is indicted on the sensorgram by a drop in RU over time as analyte dissociates from the surface-bound ligand.
A representative sensorgram for the BIAcore instrument is presented in FIG. 1, which depicts an antibody surface interacting with analyte in a sample. During sample injection, an increase in signal is observed due to binding of the analyte (i.e., association) to a steady state condition where the resonance signal plateaus. At the end of sample injection, the sample is replaced with a continuous flow of buffer and decrease in signal reflects the dissociation of analyte from the surface. The slope of the association/dissociation curves provide valuable information regarding the reaction kinetics, and the height of the resonance signal represents surface concentration (i.e., the response resulting from an interaction is related to the change in mass concentration on the surface). While dissociation will naturally tend to regenerate some portion of the sensor surface, only a very small portion of the sensor surface is typically regenerated in this manner, especially when their is a strong interaction between the ligand and analyte. Thus, some further regeneration step is often needed in order to effectively remove analyte from the sensor surface and ready the surface for contact with a new sample.
Numerous articles have been published directed to the use of the BIAcore instrument in the analysis of biomolecular interactions. In these articles, researchers have reported a variety of regeneration agents and techniques for regenerating the sensor surface prior to contact with a new sample. In general, these articles had main goals other than surface regeneration; however, three papers discussed systematic investigations of regeneration practices concerning antibody-antigen assays (Brigham & O'Shannessy, Chromatographia 35:45-49, 1993; Brigham et al., Analytical Biochemisty 205:125-131, 1992; Minunni et al., Analytical Letters 26:1441-60, 1992), with perhaps the most extensive treatment being that of Burke & O'Shannesy (1993). In that reference, a sCR1-MAb YZ1 system was regenerated using various regeneration agents. The results of this study indicated that, among several common regeneration agents, only a few had a high regeneration effect for the sCR1-MAb YZ1 system. The authors reported that the choice of acid can be more important than the choice of pH (e.g., 0.1M phosphoric acid, pH 1.3, worked better than 0.1M HCl, pH 1.0), and that combinations of agents in some cases are favorable (e.g., 50% ethyleneglycol/0.1M triethylamine, pH 10.5, was more favorable than 0.1M triethylamine, pH 10.5).
More generally, the above-noted articles disclose that various classes of ligand-analyte systems may be regenerated under the following conditions:
Antibody-antigen assays--to varying degrees with hydrochloric acid (HCl) of different concentrations (Malmborg et al, Scandinavial Journal of Immunology 35:643-50, 1992; Ward et al., Biochemistry International 26:559-65, 1992) or with weaker acids, typically phosphoric or formic (Corr et al., Journal of Experimental Medicine 178:1877-92, 1993; VanCott et al., Journal of Immunological Methods 183:103-17, 1995), or with detergent or chaotropic solutions (Tanchou et al., AIDS Research and Human Retroviruses, 10:983-93 1994; End et al., Journal of Biological Chemistry 268:10066-75, 1993);
Receptor-transmitter assays--with acids (Morelock et al., Journal of Medicinal Chemistry 38:1309-18, 1995), bases (Lemmon et al., Journal of Biological Chemistry 269:31653-58, 1994), under chaotropic conditions and high ion strength (Stitt et al., Cell 80:661-70, 1995), or under natural dissociation conditions (Ma et al., Journal of Biological Chemistry 39:24430-36, 1994);
Assays containing DNA--under very mild regeneration conditions using detergents, EDTA, or under natural dissociation conditions (Cheskis et al., Molecular Endocrinology 1996; Casasnovas Journal of Biological Chemistry 270:13216-24, 1995); and
Assays containing glycoproteins--under acid conditions or using sugar solutions (Okazaki et al., Journal of Molecular Recognition 8:95-99 1995).
While these articles disclose a variety of regeneration techniques, those techniques are system dependent and are not particularly effective beyond the parameters of the specific system reported in each paper. Thus, anytime a researcher investigates a new ligand-analyte system, a great deal of time and effort may be spent identifying regeneration conditions suitable for the system at hand, often with varying degrees of success. Accordingly, there is a need in the art for improved techniques for regenerating the surface of an affinity biosensor.
There is also a need in the art for techniques to characterize the analyte and/or ligand associated with the surface of an affinity biosensor. Such characterization can occur either prior to the regeneration of the biosensor surface (e.g., during association or dissociation) or can occur during regeneration. Further, the ability to predict structure-activity relationships ("SAR") has become an important goal in a variety of fields. For example, as the number of known protein structures has increased, researchers have tried, with limited success, to predict SAR for such proteins. In the context of monoclonal antibodies, one goal has been to design a MAb that binds specifically to a given antigen, in advance of laboratory experiments. Accordingly, a need exists for techniques that can predict SAR for new analytes and/or ligands, such as proteins, and thus characterize their activity in advance of laboratory analysis of the same.
There is also a need to characterize analytes and/or ligands with respect to changing chemical environments. For example, in developing quantitative assays for determination of vitamin concentration in food, researchers are often interested in knowing how sensitive a specific molecule, typically a MAb, is to variations in its chemical environment. An aqueous solution having a known amount of vitamin, for example, may be more sensitive than a crude sample (e.g., infant formulas, cereals, etc.) having the same concentration of vitamin. Therefore, the measured concentration of vitamin from the crude sample may be different than its true concentration. Similarly, when determining drug and/or hormone residues in animals (e.g., in urine), researchers are also often interested in knowing how sensitive a specific molecule is to variations in its chemical environment. Accordingly, a need exists for techniques that can predict the performance of a specific molecule in a crude sample. Such techniques may also be useful in developing quantitative assays.
Furthermore, there is also a need in the art to detect and characterize minor structural differences in, for example, proteins. Researchers often desire to verify that manufactured proteins have their expected structures, and are not point-mutated or post modified (i.e., by substitutions by carbohydrates, fatty acids, etc.). Accordingly, a need exists for methods useful for detecting minor structural differences in proteins.
The present invention fulfills these needs, and provides further related advantages.